The full extent of the role of peroxisomes in lipid metabolism remains unknown. We have recently made the exciting discovery that rat peroxisomes are capable of cholesterol oxidation. Under in vitro assay conditions designed to minimize mitochondrial Beta-oxidation, we have demonstrated that peroxisomes can account for over 30% of total liver cholesterol side chain oxidation (26-14C cholesterol to 14C -propionic acid). The remainder of the activity is localized in mitochondria. The specific aims of this proposal are to further characterize this newly-discovered peroxisomal pathway. Purified peroxisomal and mitochondrial fractions from rat liver will be used to assess cofactor requirements and substrate specificity; identification of bile acids produced by the peroxisomal and mitochondrial pathways will be carried out. Depending upon the identity of the bile acids and the extent to which their sterol ring is modified by peroxisomes, we will test for other possible peroxisomal enzymes that to date have been solely attributed to the microsomal and/or mitochondrial fractions. Peroxisomal proliferation, peroxisomal Beta -oxidation, and a corresponding decrease in serum triglycerides and cholesterol are known to be induced by a wide variety of structurally unrelated hypolipidemic drugs. Accordingly, we will examine in the rat and in a liver cell line the effects of several key hormones and hypolipidemic drugs upon the relative contribution of peroxisomes and mitochondria to net cholesterol oxidation. Elimination of cholesterol from the body occurs mainly in the liver either by direct cholesterol excretion into the bile or by cholesterol oxidation to bile acids. These processes may affect the homeostasis of plasma cholesterol levels. Defects in cholesterol handling may be important in the formation of atheromatous deposits, and in cholesterol-containing gallstones, stemming from changes in bile acid content and bile acid composition. Clarification of the role of peroxisomes in cholesterol oxidation will lead to a greater understanding of lipid-related diseases and/or peroxisomal metabolic disorders.